Cells were suspended with a Pasteur pipette, centrifuged onto glass microscope slides (Shandon Southern Instruments, Pittsburgh, Pa.), air dried, and fixed in methanol. cells at 37C. Spirochetes grown axenically in BSK-H medium also produced more OspC at 37C, but OspA content material was not appreciably affected by temp. Our findings show that temp, along with cultivation inside a tick cell tradition system, plays a role in the differential manifestation of OspA and enhances differential manifestation of OspC by spirochetes. The Lyme disease spirochete sensu stricto cycles in nature between small mammals and CCND2 ticks. This ability to invade and infect two physiologically quite divergent hosts entails alterations in Insulin levels modulator the outer surface protein (Osp) composition of proteins. Probably one of the most notable effects is an increase in OspC production and a decrease in OspA production in spirochetes during nymphal attachment and feeding (6, 8C10, 25, 29). This differential manifestation of OspA and OspC during tick feeding coincides with an increase in the infectivity of spirochetes for the mammalian sponsor (23, 24). OspA offers been shown to be indicated in significant amounts in most strains in tradition (2, 4, 10, 17, 32). An increase in temperature enhanced the manifestation of OspC in spirochetes cultured in BSK medium, whereas no effect on OspA was mentioned (7). The aim of this study was to compare OspA and OspC manifestation in sensu stricto cocultivated with tick (in the two tradition systems. Here we statement that temp modulates the manifestation of both OspA and OspC in cocultivated with cells. (This work is definitely part of the Ph.D. dissertation of Marygorret Obonyo.) MATERIALS AND METHODS Spirochete strains and cultivation. Strains JMNT (14, 22) and N40 (3) of sensu stricto (passage 3), previously maintained at 34C, were stored Insulin levels modulator freezing in liquid nitrogen. Before each experiment, freezing spirochetes were thawed at 37C, transferred to total BSK-H (Sigma, St. Louis, Mo.) Insulin levels modulator medium supplemented with 6.3% rabbit serum (Gibco, Grand Island, N.Y.) and 1.4% gelatin (Difco, Detroit, Mich.), and incubated at 34C for 4 days prior to cocultivation with tick cells or axenic cultivation Insulin levels modulator in BSK-H medium. tick cell collection ISE6 (18) was Insulin levels modulator used. The collection was taken care of in L15B (19) medium comprising 5% heat-inactivated fetal bovine serum (Sigma), 10% tryptose phosphate broth (Difco), and 1% lipoprotein cholesterol concentrate (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.). Cells, seeded into 24-well cells tradition plates (1 ml/well) or 25-cm2 cells tradition flasks (5 ml/flask), were cultivated at 31C until the cell denseness reached 2.5 106 cells/ml (usually by 7 days). Spirochetes, previously cultivated in BSK-H medium, were counted having a Petroff-Hausser bacterial counting chamber (Hausser Scientific, Horsham, Pa.) and were diluted in L15BS medium (13, 19) (1.25 107 borreliae/ml). The spent tick cell tradition medium (L15B) was replaced with an equal volume of spirochetes in L15BS medium (13, 19), and ethnicities were incubated at 31, 34, or 37C. For assessment, spirochetes cultured axenically were diluted to 105 spirochetes/ml in new complete BSK-H medium and were incubated at 31, 34, or 37C. Microscopic evaluation. Cells were suspended having a Pasteur pipette, centrifuged onto glass microscope slides (Shandon Southern Tools, Pittsburgh, Pa.), air flow dried, and fixed in methanol. Some slides were stained with 4% Giemsa (Gibco) remedy and others were utilized for indirect fluorescent-antibody (IFA) microscopy. For IFA microscopy, cells were incubated with monoclonal antibody (MAb) H5332 (2) (provided by Tom Schwan, Rocky Mountain Laboratories, National Institutes of Health) at 37C for 30 min. Thereafter, slides were washed once in phosphate-buffered saline (PBS) comprising 5 mM MgCl2 and were then reacted for 30 min at 37C with rhodamine-conjugated anti-mouse immunoglobulin G (Pierce, Rockford, Ill.). The slides were washed once in PBS comprising MgCl2 (5 mM) and were examined by fluorescence microscopy. SDS-PAGE and immunoblotting. Spirochetes cultivated axenically in BSK-H medium at 31C were harvested after 7 days, and those cultivated at 34 or 37C were passaged into new medium when they reached the mid-logarithmic phase of growth (4 days) and were harvested 3 days later. Spirochetes were counted having a bacterial counting chamber and were harvested by centrifugation (3,000 for 30 min at 4C). After a second wash with HBSS and a second round of centrifugation, pelleted spirochetes were resuspended in 2 sodium dodecyl sulfate (SDS) reducing buffer and were lysed by boiling for 5 min. Spirochetes cultivated with ISE6 cells in 25-cm2 cells.
Cells were suspended with a Pasteur pipette, centrifuged onto glass microscope slides (Shandon Southern Instruments, Pittsburgh, Pa